Original Research published: 03 August 2016 doi: 10.3389/fimmu.2016.00287
Leishmania infantum and Leishmania braziliensis: Differences and similarities to evade the innate immune system Sarah de Athayde Couto Falcão1,2, Tatiana M. G. Jaramillo1, Luciana G. Ferreira2, Daniela M. Bernardes1, Jaime M. Santana1 and Cecília B. F. Favali1,2* 1 Department of Cell Biology, Biology Institute, University of Brasília, Brasília, Brazil, 2 Núcleo de Medicina Tropical (NMT), University of Brasília, Brasília, Brazil
Edited by: Alexandre Morrot, Federal University of Rio de Janeiro, Brazil Reviewed by: Hira Nakhasi, US Food and Drug Administration, USA Paula Mello De Luca, Oswaldo Cruz Foundation, Brazil Celio Geraldo Freire De Lima, Federal University of Rio de Janeiro, Brazil *Correspondence: Cecília B. F. Favali [email protected] Specialty section: This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology Received: 25 April 2016 Accepted: 15 July 2016 Published: 03 August 2016 Citation: Falcão SAC, Jaramillo TMG, Ferreira LG, Bernardes DM, Santana JM and Favali CBF (2016) Leishmania infantum and Leishmania braziliensis: Differences and Similarities to Evade the Innate Immune System. Front. Immunol. 7:287. doi: 10.3389/fimmu.2016.00287
Visceral leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self-healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs) act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human DCs’ biology for better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood of healthy volunteer donors and from those infected with L. infantum or L. braziliensis for 24 h. We observed similar rates of infection (around 40%) as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expression when DCs were infected for 24 h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of DCs after 24 h. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model. Keywords: apoptosis, dendritic cells, Leishmania infantum, Leishmania braziliensis, CD209
INTRODUCTION Leishmaniasis is a vector-borne disease that commits millions of people around the world. In 2013, around 215,000 new cases were reported in the world. Of these, around 21,000 were reported in Brazil (1). Around 0.2–0.4 and 0.7–1.2 million visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), respectively, occur each year. Over 90% of new cases of VL occur in six countries all
Frontiers in Immunology | www.frontiersin.org
1
August 2016 | Volume 7 | Article 287
Falcão et al.
Dendritic Cell Regulation by Leishmania
infected with L. major showed an increase of HLA-DR, CD86, and CD40. When L. major-infected DCs were cocultured with T lymphocytes and treated with anti-CD40 ligand (CD40L), IL-12p70 and IFN-γ production decreased, concluding that IL-12p70 and IFN-γ production are CD40L-dependent (22). When CD86 (B72)−/− mice were infected with L. major, they presented a resistance, differently CD80 (B7-1)−/−, and wild-type Balb/c mice maintained its susceptibility. Moreover, CD86−/− produced more IL-4 than wild-type mice, suggesting that CD86 induced a Th2 response (23). Infection of BALB/c mice by Leishmania amazonensis led to higher accumulation of Langerhans cells, and CD4+ and CD8+ T cells were found that produced IL-4 and IL-10. Nevertheless, L. braziliensis infection induced dermal DCs accumulation, increased effectors, and activated memory CD4+ T cells and IL-4, IL-10, IFN-γ production by CD4+ and CD8+ T cells (24). Many pathogens alter DC biology, favoring its persistence in the infected host. Data in literature showed that L. amazonensis is able to inhibit human DC differentiation from monocytes (25) and several intracellular signaling pathways (26), altering DC biology and function. Data from murine models of leishmaniasis show that inhibitory pathways are also involved in disease pathogenesis. One example is the OX40L–OX40 pathway that enhances Th2 responses in L. major-infected mice (27). Working with Ox40l−/− mice, the authors observed that they were very susceptible to L. major and Leishmania mexicana. Interestingly, only lymphnode cells from L. major Ox40l−/− mice produced less IL-4 and IL-10 than Ox40l+/+. In this way, this pathway is relevant to Th2 development only in the context of L. major infection, but it does not alter L. major outcome of infection (28, 29). Other surface receptors, the programmed death ligand 1 (PD-L1) and 2 (PD-L2), were studied in susceptible mice infected with L. mexicana. PD-L1 knockout mouse demonstrated resistance, decrease of lesion size, as well as parasitic load and IL-4 production after infection. Although PD-L2 knockout mouse showed increase of lesion size, parasitic load, and antibodies production, no difference on IFN-γ and IL-4 production was observed. Both receptors play different roles in response to L. mexicana (30). Another way that pathogens have to adapt to the host is to alter cell death. It is well known that Mycobacterium tuberculosis induces cell death through apoptosis, but this event favors antigen presentation (31). Moreover, pathogens as Leishmania delay cell death as a way to survive inside host cell. For example, apoptosis of monocyte-derived dendritic cells (moDCs) induced by treatment with camptothecin was downregulated by infection with L. mexicana amastigotes, detected by annexin V binding to phosphatidylserine (32). It is not clear how the regulation of human DC biology is affected in different ways by species of Leishmania. In this way, the aim of this study is to analyze the expression of surface molecules relevant to antigen processing and presentation as well as DCs survival after interaction with L. infantum or L. braziliensis.
over the world, including Brazil. About 95% of CL cases occur in the Americas, the Mediterranean basin, and the Middle East and Central Asia (2). In the sandfly, the flagellated motile forms of Leishmania spp. called promastigotes progress through several morphological stages of differentiation, regulating the vector midgut environment (3). Finally, it becomes the non-dividing, infectious metacyclic promastigotes that are transmitted during a sandfly bite, when they are able to infect or be phagocyted by professional phagocytes as macrophages (4) and dendritic cells (DCs) (5). The parasite inside host cells becomes amastigote, a stage without an externalized flagellum that is capable of multiplication in antigen-presenting cells (6). CL is the most common clinical form of leishmaniasis and causes localized skin lesions, especially in arms and legs. In the New World, Leishmania braziliensis is able to cause from localized self-limited lesions to tissue destructive mucosal forms (7) that can worsen with age (8). On the other hand, visceral forms are caused by Leishmania infantum (9), and the disease, characterized by fever, weight loss, enlargement of the spleen and liver, and anemia, is fatal if left untreated (10). Host–parasite interactions during innate immune responses determine the fate of adaptive immunity, contributing to healing or parasite persistence in leishmaniasis (11). DCs are professional antigen-presenting cells that interact with pathogens in peripheral tissues and stimulate T lymphocytes after migration to secondary lymphoid organs (12). In the periphery, DCs are in an immature state, with high potential to perform phagocytosis through many receptors that recognize pathogen-associated molecular patterns, as DC-SIGN (CD209) (13–15). Toll-like receptors (TLRs) are also involved in innate response to Leishmania parasites. During murine Leishmania major infection, data in literature showed that TLR9 was required for the induction of IL-12 in bone marrow-derived DCs (BMDCs) by intact L. major parasites or L. major DNA. This IL-12 production was essential for early interferon-gamma expression and NK cell activation (16). After pathogen internalization, DC is able to migrate from periphery to secondary tissues, rising MHC expression as well as other costimulatory molecules CD86 and CD83. After all these events, DCs are able to properly perform antigen presentation to T cells and determinate the fate of adaptive immune response (17). DCs can also become tolerogenic cells, as observed in the physiologic regulation of apoptosis (18). When L. braziliensis were in contact with murine CD11c+ DCs, they were able to produce high levels of IL-12p70 as well as stimulate a significant expression of CD40 and CD83 in the surface of these DCs (19). Another study from the literature, working with mice BMDCs, showed that L. infantum could infect and survive inside these cells. Besides, they observed that L. infantum promastigotes were not able to upregulate CD40 and CD86 surface expression. The authors also observed that L. infantum was able to induce some level of IL-12p40 and IL-10, with no differences in TNF-α levels (20). Another group showed that bystander BMDCs from Balb/c mice increased IL-12p40 and expressed more CD40, CD86, and MHC class II in the cell surface than infected or not exposed cells. These bystander DCs induced a protective CD4+ IFN-γ T cells response, while L. infantuminfected DCs polarizing to T-bet+IFN-γ+IL-10+ double producer T cells phenotype (21). On the other hand, human DCs
Frontiers in Immunology | www.frontiersin.org
MATERIALS AND METHODS Parasites
Leishmania infantum (MHOM/BR/1974/PP75) and L. braziliensis (MHOM/BR/01/BA788) promastigotes were maintained
2
August 2016 | Volume 7 | Article 287
Falcão et al.
Dendritic Cell Regulation by Leishmania
in Schneider’s medium (SIGMA) with 20% of calf fetal serum (GIBCO) and gentamicin (50 μm/mL). Leishmania stationaryphase promastigotes were quantified in Neubauer chamber. After counting, promastigotes were harvested from culture bottles, washed three times with cold PBS (800 g for 10 min), and adjusted in RPMI medium to be added in DC culture.
in 190 μL of binding buffer, added 10 μL propidium iodide (PI) (annexin V and propidium iodide kit, eBioscience), and acquired on a flow cytometer. Stained cells were acquired on a Verse flow cytometer (BD Bioscience) and analyzed using FlowJo Software (Tree Star, Inc.).
Cytokine Assay
Dendritic Cell Culture
Dendritic cells were infected with both Leishmania species. After 24 h of infection, supernatant was collected and level of TNF-α was measured by ELISA. TNF-α was quantified (test detection range from 20 to 1000 pg/mL) with purified antihuman TNF-α, antihuman TNF-α biotin, and streptavidin–alkaline phosphatase, according to the instruction of suppliers (Novex, Life Technologies, Invitrogen). Absorbance was measured and analyzed with the program SoftMax Pro (Molecular Devices).
Human DCs were differentiated from monocytes. Briefly, venous blood was collected from volunteer healthy donors (n = 8), and peripheral blood mononuclear cells (PBMC) were obtained by passage over a Ficoll gradient (GE Healthcare). Cells were harvested, washed, and stained with antibodies, anti-CD14, conjugated with microbeads (Miltenyi Biotec). Stained cells (monocytes) were purified after positive selection in a magnetic field. Monocytes were then counted and cultivated in RPMI 1640 medium supplemented with 2 mM l-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin (GIBCO), IL-4 (800 IU/mL), and GM-CSF (50 μg/mL) (both from PeproTech) with 10% of fetal serum bovine (GIBCO) in 24-wells plates (5 × 105 cells/wells) for 7 days at 37°C and 5% CO2. New medium (200 μL) supplemented with cytokines was added on days 3 and 6 of the culture. On day 7, DCs were infected (1 DC:10 parasites), cultivated in 24-wells plates for 24 h at 37°C and 5% CO2, harvested, and characterized by flow cytometry (FACS Verse BD Biosciences). All experimental conditions and measurements were performed with the same donors.
Ethical Statement
Human blood samples were collected after the signature of an informed consent signed by all volunteers, and the project was approved by The Ethical Committee for Human Beings from the Medicine Faculty of University of Brasilia (approval no. 072/2009). Samples were collected by venous puncture by a trained and specialized laboratory technician in the Universidade de Brasília.
Statistical Analysis
The statistical analysis was conducted using GraphPad Prism Software. Samples were tested by Shapiro–Wilk normality test, and the significance of the results was calculated using parametrical paired t test, and a p-value of
Leishmania infantum and Leishmania braziliensis: Differences and similarities to evade the innate immune system Sarah de Athayde Couto Falcão1,2, Tatiana M. G. Jaramillo1, Luciana G. Ferreira2, Daniela M. Bernardes1, Jaime M. Santana1 and Cecília B. F. Favali1,2* 1 Department of Cell Biology, Biology Institute, University of Brasília, Brasília, Brazil, 2 Núcleo de Medicina Tropical (NMT), University of Brasília, Brasília, Brazil
Edited by: Alexandre Morrot, Federal University of Rio de Janeiro, Brazil Reviewed by: Hira Nakhasi, US Food and Drug Administration, USA Paula Mello De Luca, Oswaldo Cruz Foundation, Brazil Celio Geraldo Freire De Lima, Federal University of Rio de Janeiro, Brazil *Correspondence: Cecília B. F. Favali [email protected] Specialty section: This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology Received: 25 April 2016 Accepted: 15 July 2016 Published: 03 August 2016 Citation: Falcão SAC, Jaramillo TMG, Ferreira LG, Bernardes DM, Santana JM and Favali CBF (2016) Leishmania infantum and Leishmania braziliensis: Differences and Similarities to Evade the Innate Immune System. Front. Immunol. 7:287. doi: 10.3389/fimmu.2016.00287
Visceral leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self-healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs) act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human DCs’ biology for better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood of healthy volunteer donors and from those infected with L. infantum or L. braziliensis for 24 h. We observed similar rates of infection (around 40%) as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expression when DCs were infected for 24 h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of DCs after 24 h. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model. Keywords: apoptosis, dendritic cells, Leishmania infantum, Leishmania braziliensis, CD209
INTRODUCTION Leishmaniasis is a vector-borne disease that commits millions of people around the world. In 2013, around 215,000 new cases were reported in the world. Of these, around 21,000 were reported in Brazil (1). Around 0.2–0.4 and 0.7–1.2 million visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL), respectively, occur each year. Over 90% of new cases of VL occur in six countries all
Frontiers in Immunology | www.frontiersin.org
1
August 2016 | Volume 7 | Article 287
Falcão et al.
Dendritic Cell Regulation by Leishmania
infected with L. major showed an increase of HLA-DR, CD86, and CD40. When L. major-infected DCs were cocultured with T lymphocytes and treated with anti-CD40 ligand (CD40L), IL-12p70 and IFN-γ production decreased, concluding that IL-12p70 and IFN-γ production are CD40L-dependent (22). When CD86 (B72)−/− mice were infected with L. major, they presented a resistance, differently CD80 (B7-1)−/−, and wild-type Balb/c mice maintained its susceptibility. Moreover, CD86−/− produced more IL-4 than wild-type mice, suggesting that CD86 induced a Th2 response (23). Infection of BALB/c mice by Leishmania amazonensis led to higher accumulation of Langerhans cells, and CD4+ and CD8+ T cells were found that produced IL-4 and IL-10. Nevertheless, L. braziliensis infection induced dermal DCs accumulation, increased effectors, and activated memory CD4+ T cells and IL-4, IL-10, IFN-γ production by CD4+ and CD8+ T cells (24). Many pathogens alter DC biology, favoring its persistence in the infected host. Data in literature showed that L. amazonensis is able to inhibit human DC differentiation from monocytes (25) and several intracellular signaling pathways (26), altering DC biology and function. Data from murine models of leishmaniasis show that inhibitory pathways are also involved in disease pathogenesis. One example is the OX40L–OX40 pathway that enhances Th2 responses in L. major-infected mice (27). Working with Ox40l−/− mice, the authors observed that they were very susceptible to L. major and Leishmania mexicana. Interestingly, only lymphnode cells from L. major Ox40l−/− mice produced less IL-4 and IL-10 than Ox40l+/+. In this way, this pathway is relevant to Th2 development only in the context of L. major infection, but it does not alter L. major outcome of infection (28, 29). Other surface receptors, the programmed death ligand 1 (PD-L1) and 2 (PD-L2), were studied in susceptible mice infected with L. mexicana. PD-L1 knockout mouse demonstrated resistance, decrease of lesion size, as well as parasitic load and IL-4 production after infection. Although PD-L2 knockout mouse showed increase of lesion size, parasitic load, and antibodies production, no difference on IFN-γ and IL-4 production was observed. Both receptors play different roles in response to L. mexicana (30). Another way that pathogens have to adapt to the host is to alter cell death. It is well known that Mycobacterium tuberculosis induces cell death through apoptosis, but this event favors antigen presentation (31). Moreover, pathogens as Leishmania delay cell death as a way to survive inside host cell. For example, apoptosis of monocyte-derived dendritic cells (moDCs) induced by treatment with camptothecin was downregulated by infection with L. mexicana amastigotes, detected by annexin V binding to phosphatidylserine (32). It is not clear how the regulation of human DC biology is affected in different ways by species of Leishmania. In this way, the aim of this study is to analyze the expression of surface molecules relevant to antigen processing and presentation as well as DCs survival after interaction with L. infantum or L. braziliensis.
over the world, including Brazil. About 95% of CL cases occur in the Americas, the Mediterranean basin, and the Middle East and Central Asia (2). In the sandfly, the flagellated motile forms of Leishmania spp. called promastigotes progress through several morphological stages of differentiation, regulating the vector midgut environment (3). Finally, it becomes the non-dividing, infectious metacyclic promastigotes that are transmitted during a sandfly bite, when they are able to infect or be phagocyted by professional phagocytes as macrophages (4) and dendritic cells (DCs) (5). The parasite inside host cells becomes amastigote, a stage without an externalized flagellum that is capable of multiplication in antigen-presenting cells (6). CL is the most common clinical form of leishmaniasis and causes localized skin lesions, especially in arms and legs. In the New World, Leishmania braziliensis is able to cause from localized self-limited lesions to tissue destructive mucosal forms (7) that can worsen with age (8). On the other hand, visceral forms are caused by Leishmania infantum (9), and the disease, characterized by fever, weight loss, enlargement of the spleen and liver, and anemia, is fatal if left untreated (10). Host–parasite interactions during innate immune responses determine the fate of adaptive immunity, contributing to healing or parasite persistence in leishmaniasis (11). DCs are professional antigen-presenting cells that interact with pathogens in peripheral tissues and stimulate T lymphocytes after migration to secondary lymphoid organs (12). In the periphery, DCs are in an immature state, with high potential to perform phagocytosis through many receptors that recognize pathogen-associated molecular patterns, as DC-SIGN (CD209) (13–15). Toll-like receptors (TLRs) are also involved in innate response to Leishmania parasites. During murine Leishmania major infection, data in literature showed that TLR9 was required for the induction of IL-12 in bone marrow-derived DCs (BMDCs) by intact L. major parasites or L. major DNA. This IL-12 production was essential for early interferon-gamma expression and NK cell activation (16). After pathogen internalization, DC is able to migrate from periphery to secondary tissues, rising MHC expression as well as other costimulatory molecules CD86 and CD83. After all these events, DCs are able to properly perform antigen presentation to T cells and determinate the fate of adaptive immune response (17). DCs can also become tolerogenic cells, as observed in the physiologic regulation of apoptosis (18). When L. braziliensis were in contact with murine CD11c+ DCs, they were able to produce high levels of IL-12p70 as well as stimulate a significant expression of CD40 and CD83 in the surface of these DCs (19). Another study from the literature, working with mice BMDCs, showed that L. infantum could infect and survive inside these cells. Besides, they observed that L. infantum promastigotes were not able to upregulate CD40 and CD86 surface expression. The authors also observed that L. infantum was able to induce some level of IL-12p40 and IL-10, with no differences in TNF-α levels (20). Another group showed that bystander BMDCs from Balb/c mice increased IL-12p40 and expressed more CD40, CD86, and MHC class II in the cell surface than infected or not exposed cells. These bystander DCs induced a protective CD4+ IFN-γ T cells response, while L. infantuminfected DCs polarizing to T-bet+IFN-γ+IL-10+ double producer T cells phenotype (21). On the other hand, human DCs
Frontiers in Immunology | www.frontiersin.org
MATERIALS AND METHODS Parasites
Leishmania infantum (MHOM/BR/1974/PP75) and L. braziliensis (MHOM/BR/01/BA788) promastigotes were maintained
2
August 2016 | Volume 7 | Article 287
Falcão et al.
Dendritic Cell Regulation by Leishmania
in Schneider’s medium (SIGMA) with 20% of calf fetal serum (GIBCO) and gentamicin (50 μm/mL). Leishmania stationaryphase promastigotes were quantified in Neubauer chamber. After counting, promastigotes were harvested from culture bottles, washed three times with cold PBS (800 g for 10 min), and adjusted in RPMI medium to be added in DC culture.
in 190 μL of binding buffer, added 10 μL propidium iodide (PI) (annexin V and propidium iodide kit, eBioscience), and acquired on a flow cytometer. Stained cells were acquired on a Verse flow cytometer (BD Bioscience) and analyzed using FlowJo Software (Tree Star, Inc.).
Cytokine Assay
Dendritic Cell Culture
Dendritic cells were infected with both Leishmania species. After 24 h of infection, supernatant was collected and level of TNF-α was measured by ELISA. TNF-α was quantified (test detection range from 20 to 1000 pg/mL) with purified antihuman TNF-α, antihuman TNF-α biotin, and streptavidin–alkaline phosphatase, according to the instruction of suppliers (Novex, Life Technologies, Invitrogen). Absorbance was measured and analyzed with the program SoftMax Pro (Molecular Devices).
Human DCs were differentiated from monocytes. Briefly, venous blood was collected from volunteer healthy donors (n = 8), and peripheral blood mononuclear cells (PBMC) were obtained by passage over a Ficoll gradient (GE Healthcare). Cells were harvested, washed, and stained with antibodies, anti-CD14, conjugated with microbeads (Miltenyi Biotec). Stained cells (monocytes) were purified after positive selection in a magnetic field. Monocytes were then counted and cultivated in RPMI 1640 medium supplemented with 2 mM l-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin (GIBCO), IL-4 (800 IU/mL), and GM-CSF (50 μg/mL) (both from PeproTech) with 10% of fetal serum bovine (GIBCO) in 24-wells plates (5 × 105 cells/wells) for 7 days at 37°C and 5% CO2. New medium (200 μL) supplemented with cytokines was added on days 3 and 6 of the culture. On day 7, DCs were infected (1 DC:10 parasites), cultivated in 24-wells plates for 24 h at 37°C and 5% CO2, harvested, and characterized by flow cytometry (FACS Verse BD Biosciences). All experimental conditions and measurements were performed with the same donors.
Ethical Statement
Human blood samples were collected after the signature of an informed consent signed by all volunteers, and the project was approved by The Ethical Committee for Human Beings from the Medicine Faculty of University of Brasilia (approval no. 072/2009). Samples were collected by venous puncture by a trained and specialized laboratory technician in the Universidade de Brasília.
Statistical Analysis
The statistical analysis was conducted using GraphPad Prism Software. Samples were tested by Shapiro–Wilk normality test, and the significance of the results was calculated using parametrical paired t test, and a p-value of
